Automated in vivo compound screening with zebrafish and the discovery and validation of PD 81,723 as a novel angiogenesis inhibitor

Angiogenesis is a critical process in tumor progression. Inhibition of angiogenesis by blocking VEGF signaling can impair existing tumor vessels and halt tumor progression. However, the benefits are transient, and most patients who initially respond to these therapies develop resistance. Accordingly, there is a need for new anti-angiogenesis therapeutics to delay the processes of resistance or eliminate the resistive effects entirely. This manuscript presents the results of a screen of the National Institutes of Health Clinical Collections Libraries I & II (NIHCCLI&II) for novel angiogenesis inhibitors. The 727 compounds of the NIHCCLI&II library were screened with a high-throughput drug discovery platform (HTP) developed previously with angiogenesis-specific protocols utilizing zebrafish. The screen resulted in 14 hit compounds that were subsequently narrowed down to one, with PD 81,723 chosen as the lead compound. PD 81,723 was validated as an inhibitor of angiogenesis in vivo in zebrafish and in vitro in human umbilical vein endothelial cells (HUVECs). Zebrafish exposed to PD 81,723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemical analysis did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured HUVECs elucidated the ability of PD 81,723 to inhibit capillary tube formation, migration, and proliferation of endothelial cells. In addition, PD 81,723 did not induce apoptosis while significantly down regulating p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A in cultured HUVECs.

www.nature.com/scientificreports/ excessive, and/or abnormal angiogenesis contributes to the pathogenesis of many disorders, such as cancer [2][3][4][5] , rheumatoid arthritis 6 , psoriasis 7 , and diabetic retinopathy 8 . In disorders featuring excessive angiogenesis, there is a surplus of pro-angiogenesis factors that may be accompanied by a reduction of angiogenesis inhibitors. Cancer is the most prominent and most studied disease in this category. Tumor progression is dependent on angiogenesis, as uncontrollable cell growth and metastasis cannot be sustained in the absence of neovascularisation 9 . The idea of targeting pathological angiogenesis to starve tumors was introduced nearly 50 years ago and termed anti-angiogenesis 10 . Several anti-angiogenesis therapies, mainly targeting the VEGF-A/VEGFR-2 signalling axis, have been approved as possible therapeutics for a number of tumor types. However, the benefits are transient, and the vast majority of patients who initially respond to the therapies develop resistance with higher levels of tumor invasiveness and the formation of distant metastasis 11,12 . This indicates a need for new anti-angiogenesis therapeutics that can delay the processes of resistance or eliminate the resistive effects entirely.
The popularity of zebrafish as a well-managed vertebrate model to study human diseases is due to its unique combination of optical clarity as embryos and larvae, and embryological manipulability, which allows for the examination of the onset and progression of a pathological process in vivo and in real time 13 .
The goal of maximizing the high-throughput potential of zebrafish led to the construction and functional development of a high-throughput drug discovery platform (HTP), with the capacity to perform fully automated screens of compound libraries using zebrafish embryos 14 . The pilot project for this prototypical system, in which the National Institutes of Health Clinical Collections Libraries I & II (NIHCCLI&II) were screened for angiogenesis inhibitors, resulted in the discovery of PD 81,723 as a potential angiogenesis inhibitor. This manuscript is a report on the results of this pilot project and the validation of PD 81,723 as an inhibitor of angiogenesis in vivo in zebrafish and in vitro in cultured endothelial cells.

Screening the NIH clinical collections libraries I & II.
There were 14 hit compounds from the screen of the NIHCCLI&II libraries for angiogenesis inhibitors. The library was screened 3 times with the HTP protocol described previously 14 at a concentration of 5 μM for each compound. The compounds were introduced to the embryos at 10-11 h post-fertilization (hpf) and the readouts were taken at 4 days post-fertilization (dpf). The hit compounds are shown in Table 1, ranked according to the standard deviation from the 3 trials. Representative images of the hit compounds, with the pixels containing endothelial cells identified and enhanced, were compared and the 5 compounds that corresponded to the most diminished endothelial network with a relatively normal overall morphology were chosen for further analysis. The representative images of all the hit compounds can be found in Supplementary Fig. S1, with the top 5 hits highlighted. The top 5 compounds were droperidol, mesoridazine, metaproterenol, PD 81,723, and raclopride. Figure 1A shows the representative images of Tg(kdrl:EGFP) zebrafish treated with the top 5 hit compounds compared to a control dosed with 0.05% dimethyl sulfoxide (DMSO).
Evaluating the top 5 hits. An experiment using the anti-angiogenesis HTP protocol was conducted, with multiple concentrations of each of the top 5 compounds, to determine which compounds would yield a significant result with a higher number of samples. Tg(kdrl:EGFP) zebrafish were distributed 1 fish/well and placed in a 96-well plate, with two columns (N = 16) designated for each concentration. The concentrations of the compounds were 0 µM (vehicle; 0.05% DMSO or Milli-Q water), 1, 2, 4, 8, and 16 µM and the experiment Table 1. The 14 hit compounds from a screen of the NIHCCLI&II libraries for angiogenesis inhibitors. The library was screened three times. Compounds were considered hits if they reduced the endothelial network by 30% or more on average when compared to the average of the negative control endothelial network. More value was given to consistency; therefore, the compounds were ranked according to the standard deviation between the three replicates.

Rank
Compound % of negative control mean Standard deviation (%)  1B). None of the compounds seemed to have a major impact on mortality in these experiments (Fig. 1C).
Selection of PD 81,723. Due to their ability to significantly reduce the zebrafish endothelial network, PD 81,723 and Raclopride were evaluated further at the concentrations that resulted in a statistically significant result. Fluorescent images were taken at 1, 2, 3, and 4 dpf to monitor the chemical phenotypes due to PD 81,723 (at 4, 8, and 16 µM) and raclopride (at 16 µM). (Supplementary Fig. S2). The fish exposed to PD 81,723 and raclopride exhibited features, such as a hindrance in the development of the caudal vein and artery, diminished EGFP expression in the intersegmental vessels (ISVs) and cranial vasculature, and cardiac edema, that are often observed in the presence of an angiogenesis inhibitor [15][16][17] . These features were not present in zebrafish dosed with 16 µM raclopride at 3 dpf and 4 dpf. They were only present in the form of a diminished EGFP signal coming from the ISVs in zebrafish dosed with 16 µM PD 81,723. For this reason, in addition to a variable affect that seemed independent to concentration (see Fig. 1B), raclopride was excluded from further study. Since, PD 81,723 appeared to be working in a dose-dependent manner; a dose response experiment was conducted with higher concentrations of this compound in order to find the highest concentration that could be used without severely impacting survival. The concentrations of PD 81,723 used were 0 µM (vehicle control; 0.05% DMSO), 16,32,64,128, and 256 µM (N = 16/dose). This experiment was repeated in triplicate using the protocol mentioned previously 14 , with survival as the only readout. This experiment yielded an LD50 concentration of 73.28 µM. With this in mind, 64 µM was chosen as the working concentration of PD 81,723 for further evaluation. Fluorescent images were taken at 1, 2, 3, and 4 dpf to monitor the chemical phenotypes due to PD 81,723 at 64 µM. Figure 2 shows the representative images from this experiment. The fish in this experiment dosed with PD 81,723 at 64 µM exhibited features that are often seen in the presence of an angiogenesis inhibitor that were still evident at the 4 dpf mark. In addition to the diminished EGFP expression in the ISVs and cranial vasculature and cardiac edema, the sub-intestinal vessels (SIVs) were missing in the fish dosed with 64 µM of PD 81,723. Figure 2B highlights the SIV region in the 4 dpf images from Fig. 2A. SIV development has been used in the evaluation of angiogenesis modulators in the past 17 .

Validation of PD 81,723 as an angiogenesis inhibitor in zebrafish.
To confirm the inhibitory effects of PD 81,723 and build a hypothesis for a possible mechanism of action, confocal imaging was employed to construct 3D images of zebrafish at 4 dpf dosed with 64 µM PD 81,723 at 10-11 hpf. Whole body and cranial end images were taken of Tg(kdrl:EGFP) and Tg(Fli1:nEGFP) zebrafish ( Supplementary Fig. S3). Images of the whole Tg(kdrl:EGFP) fish body showed results similar to the 2D fluorescent images. They exhibited a diminished EGFP signal coming from the ISVs and cranial vasculature and smaller malformed SIVs when compared to the control. The images on the cranial end of the Tg(kdrl:EGFP) fish, focused on the area around the SIVs, showed a missing vertebral artery (VTA) and parachordal vessel (PAV), a reduction in the vasculature were the ISVs meet the posterior cardinal vein (PCV), and a smaller and malformed SIV network. Images of the whole Tg(fli1:nEGFP) fish body did not show a diminished EGFP signal coming from the ISVs and cranial vasculature as in the Tg(kdrl:EGFP) fish, but smaller malformed SIVs were evident when compared to the control. The www.nature.com/scientificreports/ images on the cranial end of the Tg(fli1:nEGFP) fish showed nuclei that appeared much larger and less numerous in the PD 81,723 dosed fish when compared to the control. Based on results of the 3D confocal imaging, the area surrounding the SIVs and the ocular vasculature were selected as the regions of interest for the remaining zebrafish experiments. To quantify the inhibitory effect of PD 81,723 on angiogenesis, 3D confocal imaging focusing on the SIVs area was done with a larger sample size (N = 3 repeated three times), using Tg(kdrl:EGFP) fish. The number of vessels sprouting from the base of the SIVs basket was counted and the presence (or absence) of complete VTA and PAV was observed. There was a significant decrease in the mean number of vessels sprouting from the SIVs basket in the fish dosed with 64 µM of PD 81,723 when compared to the control (P < 0.05 vs. control). Only 22% of the fish dosed with PD 81,723 demonstrated complete VTA and PAV, compared to 89% in the control (Fig. 3).
In order to look at the functionality of the SIVs, high framerate time-lapse images of Tg(kdrl:EGFP;GATA-1:DsRed) double transgenic zebrafish were taken with a spinning disc confocal microscope to observe and evaluate blood flow. The erythroid-specific GATA-1 promoter is directing DsRed expression in red blood cells. The resulting flow videos (Supplementary Video S1) showed a significant amount of flow in the base vessel of the SIVs basket and a complete absence of flow in vessels sprouting from the base at the caudal end in both the control and PD 81,723 dosed fish. The flow in vessels sprouting from the base at the rostral end was less www.nature.com/scientificreports/ significant in the PD 81,723 dosed fish, when compared to the control. However, this may have been due to the underdeveloped nature of the SIVs in the PD 81,723 fish. Since PD 81,723 seemed to impact the proliferation of endothelial cells, immunochemistry experiments were performed to look at the effect of PD 81,723 on the apoptotic and mitotic activity in the regions of interest (ocular vasculature and SIVs). Tg(fli1:nEGFP) fish were fixed with paraformaldehyde (PFA) and tagged with antibodies targeted against active caspase-3 and phosphorylated histone H3 (p-Histone H3), an apoptotic and mitotic marker, respectively. The ocular vasculature and SIVs were imaged for each fish (N = 3 repeated three times). The number of endothelial nuclei was significantly reduced in both regions of interest in all instances of this experiment when comparing the means (P < 0.05 or P < 0.01 vs. control). Significant apoptotic activity was not observed in the tissue surrounding the ocular vasculature or the SIVs in PD 81,723 fish, when compared to the control group (Fig. 4). A significant reduction in the mean mitotic activity in the tissue surrounding the ocular vasculature and the SIVs in PD 81,723 fish was observed, when compared to the control group (P < 0.01 and P < 0.05 respectively vs. control) (Fig. 5). As anticipated, there was no colocalization observed with the apoptotic marker and the endothelial nuclei because the dying cells would have a very weak (or absent) EGFP signal. Unexpectedly, colocalization was not observed with the mitotic marker and the endothelial nuclei.

Validation of PD 81,723 as an angiogenesis inhibitor in HUVECs.
To determine the modulatory effect of PD 81,723 on the endothelial cell phenotype, widely accepted assays of endothelial cell function were conducted, namely matrigel tube formation, wound healing/cell migration, and cell proliferation assays 18,19 . For the matrigel assay, growth factor reduced matrigel was plated onto 96-well plates and seeded with human umbilical vein endothelial cells (HUVECs). Wells were treated with a vehicle control (0.05% DMSO) or 50 µM PD 81,723. Six 1 mm 2 sites from the center of each well were analyzed for total capillary tube length at 9 h postseeding. The wells treated with PD 81,723 exhibited a significantly lower average number of capillary tubes when compared to the control (P < 0.001) (Fig. 6A).
In the wound healing assay, HUVECs were treated with a control (0.05% DMSO) or 50 µM PD 81,723, in either complete endothelial growth media or endothelial growth media without VEGF-A, with three wells for each condition. The wounds treated with PD 81,723 in complete media and DMSO in media without VEGF-A    www.nature.com/scientificreports/ took significantly longer to close than the wounds treated with DMSO in complete media (P < 0.05) (Fig. 6B). The wounds treated with PD 81,723 in VEGF-A deficient media did not close within a 24 h period. Supplementary Video S2 exhibits time-lapse videos representative of each condition over the 24 h. A bromodeoxyuridine (BrdU) incorporation cell proliferation analysis did not reveal any notable difference in either group treated with a control, while PD 81,723 significantly reduced cell proliferation in both cases when comparing the means from three replicates (P < 0.01 vs. vehicle control in complete media) (Fig. 6C).
An annexin V/propidium iodide (PI) flow cytometry assay was performed to assess apoptotic activity in HUVECs treated with PD 81,723. Figure 7A presents a representative scatter plot divided into quadrants showing the distribution of cells marked with annexin V, PI, and double stained with annexin V and PI. Figure 7B is a bar graph exhibiting the amount of apoptotic activity for each condition. There was no significant change in the levels of apoptotic events in PD 81,723 treated cells, independent of the addition of VEGF-A to the growth media.
In addition, immunoblotting was performed to quantify the levels of key proteins known to play a role in endothelial function, such as p21, VEGF-A, AKT, phosphorylated AKT (p-AKT), VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS. PD 81,723 treated cells expressed significantly lower levels of p21, independent of the addition of VEGF-A in the growth media (P < 0.001 vs vehicle control in complete media). Likewise, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS did not present any detectable bands, indicating reduced expression in PD 81,723 treated cells. There was no statistically significant change in endogenous VEGF-A mean protein levels in PD 81,723 treated cells. AKT was decreased in the PD 81,723 treated cells, with a statistically significant decrease in PD 81,723 treated cells in complete media (P < 0.05 vs vehicle control in complete media). While, p-AKT appeared unchanged, the ratio of p-AKT/AKT was increased in PD 81,723 treated cells; with a statistically significant increase in PD 81,723 treated cells in VEGF-A deficient media (P < 0.05 vs vehicle control in complete media) (Fig. 8).

Discussion
The screen of the NIHCCLI&II in this work is a phenotype based screen in which the size of the fluorescent vascular network of Tg(kdrl:EGFP) zebrafish (Danio rerio) is quantified in the presence of various compounds. Most small molecule screens in zebrafish are phenotype-based screens, with more than 70 of such screens reported in literature 20,21 . The superiority of the phenotype-based approach is due to several factors: (1) A phenotypic screen can find a compound that can exhibit its efficacy without a validated target. (2) The therapeutic effect of a lead compound from a phenotypic screen can be due to activity at multiple targets. (3) Phenotypic screens can also definitively exclude compounds that cause undesirable outcomes, such as those of highly toxic compounds. Zebrafish screens are typically performed in living embryos or larvae which possess fully integrated vertebrate organ systems, such as functional livers, kidneys, and blood-brain barriers [22][23][24] . This can provide insight into pharmacological characteristics of the compounds being screened, such as absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox). To invoke or modulate an in vivo phenotype in zebrafish assays, compounds must inherently be absorbed and reach the target tissue, without being rapidly metabolized and/or excreted. This means that compounds discovered in zebrafish screens can be more readily translated to in vivo mammalian models without the extensive optimization of their pharmacological properties 20 . This conserved drug metabolism and ability to evaluate general and organ specific toxicity has been reviewed in the past 25 .
This report presents the discovery of PD 81,723 as potential angiogenesis inhibitor via a fully automated screen of the 727 compounds in the NIHCCLI&II library with subsequent validation in vivo with zebrafish and in vitro with HUVECs. This represents the completion of a successful pilot project for the HTP and serves as further validation for the methods developed for this screen 14 . Zebrafish are ideal for this type of large scale high-throughput screening for chemical and genetic phenotypes because of their small size, optical clarity as embryos and larvae, embryological manipulability, and high fecundity. The strong and definitive fluorescent endothelial cells in the Tg(kdrl:EGFP) fish used in the screens were instrumental in defining and quantifying chemical phenotypes. The lateral symmetry of the endothelial network and strong fluorescent signal of the fish facilitated the quantification of the network and in turn the amount of angiogenesis. www.nature.com/scientificreports/ PD 81,723 has been proven as an angiogenesis inhibitor in vivo in zebrafish and in vitro in cultured HUVECs. Tg(kdrl:EGFP) and Tg(Fli1:nEGFP) exposed to PD 81,723 exhibited features that are often seen in the presence of an angiogenesis inhibitor in addition to a reduction in the size and complexity of the overall vascular network, such as a hindrance in the development of the caudal vein and artery, diminished EGFP expression in the ISVs and cranial vasculature, cardiac edema, and a smaller SIVs network. In addition to this, detailed 3D imaging in conjunction with immunochemistry experiments revealed a lower count of endothelial cells and a lack of apoptotic and mitotic activity in the region of those cells when the zebrafish were exposed to PD 81,723. As www.nature.com/scientificreports/ anticipated, there was no colocalization observed with the apoptotic marker and the endothelial nuclei because the dying cells would have a very weak (or absent) EGFP signal. Unexpectedly, colocalization was not observed with the mitotic marker and the endothelial nuclei. It could be inferred that the reduction in mitotic activity in surrounding tissue would likely affect the rate of angiogenesis because angiogenesis signaling is exogenous in nature and proportional to the demands of surrounding tissue. However, nothing could be inferred about the direct impact of PD 81,723 on endothelial cells. This led to the next step, where cultured HUVECs were used to observe the direct effect of PD 81,723 on endothelial cells. HUVECs exposed to PD 81,723 resulted in complimentary results. The main indices of endothelial function (angiogenic potential, migration, and cell proliferation) were significantly reduced in PD 81,723-treated HUVECs when compared to a vehicle control. In addition, an annexin V/PI flow cytometry assay did not reveal significant levels of apoptotic events in PD 81,723-treated cells, independent of the addition of VEGF-A in the growth media. These results fall in line with the immunochemistry experiments looking at active caspase-3 as the apoptotic marker in zebrafish (Fig. 4).
In addition, immunoblotting revealed that PD 81,723 significantly down regulated p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A. PD 81,723 treated cells expressed significantly lower levels of p21, independent of the addition of VEGF-A in the growth media. This was not expected because p21 is reported to be downregulated in proliferating cells 26 . However, in a recent study endothelial cells were shown to express p21 to a greater degree in migrating tips cells where there is a higher level of VEGFR-2 activity 27 . On the other hand, stalk cells with less VEGFR-2 activity and higher Dll4/Notch activity coincided with cell cycle arrest and lower levels of p21 27,28 . This falls in line with the undetectable quantities of VEGFR-2 and p-VEGFR-2 observed in PD 81,723 treated cells. But, given the context-dependant role of p21 in several complex proliferative and non-proliferative pathways, the precise effect of PD 81,723 on p21 expression and its role needs further investigation 29 . There was no significant change in endogenous VEGF-A mean protein levels in PD 81,723 treated cells, which would lay the blame of a malfunctioning VEGF-A/VEGFR-2 signalling cascade on a lack of expression of VEGFR-2. AKT was decreased and p-AKT/AKT was increased in PD 81,723 treated cells relative to the DMSO control in complete media. A major role of AKT in endothelial cells is as a mediator of cell function by phosphorylating eNOS, leading to the production of nitric oxide (NO), which can promote vasodilation, angiogenesis, and cell proliferation 30,31 . The down regulation of AKT may have been nullified by relative upregulation of its phosphorylation.
The results indicate that PD 81,723 may be driving endothelial cells into a quiescent state. Cellular quiescence is a non-proliferating state that is reversible 32 . Generally endothelial cells are kept in a quiescent state in the relative absence of pro-angiogenesis stimuli, with low levels of autocrine VEGF-A signalling helping maintain endothelial cell homeostasis 1 . With VEGFR-2 being significantly downregulated and no significant change in endogenous VEGF-A, the VEGF signalling required to promote a pro-angiogenesis environment is not achievable, but the low levels required for homeostasis may be achievable in the presence of PD 81,723. In addition, the low levels of p21 and lack of additional cell death may also be indicative of cell cycle arrest. This could be significant for cancer treatments, as dormant/quiescent endothelial cells can help keep tumors dormant and inhibit their growth 33,34 .
PD 81,723 is an allosteric potentiator selectively acting at the adenosine A 1 receptor via agonist-dependent and -independent mechanisms. It is thought to enhance an agonist affinity for A 1 receptors and increase the half-life (t ½ ) of dissociation from them [35][36][37][38] . PD 81 723 was one of the key compounds originally identified as an allosteric regulator of A 1 receptor binding by the Parke-Davis Pharmaceutical Research Division 36,37 . The original compound series was identified from a 300-ligand adenosine A 1 binding screen of the Parke-Davis compound bank. The 2-amino-3-benzoylthiophene chemical scaffolds were originally synthesized as intermediates for benzodiazepine-like compounds, but the recognition of their ability to modulate adenosine prompted further analysis. Second generation compounds from this study were found to increase the specific binding of [ 3 H]N 6 -cyclohexyladenosine, a selective A 1 receptor agonist, to rat brain membranes. As a result, further compounds were synthesized to identify essential structure-activity relationships and PD 81,723 was identified as an analogue with an improved allosteric profile [35][36][37][38] .
There have been many studies that associate allosteric potentiators of adenosine receptors and adenosine itself with pro-angiogenesis and mitogenic effects [39][40][41][42] . There have also been studies that associate elevated levels of adenosine and adenosine agonists with endothelial cell apoptosis 43,44 . With several other studies citing the ability of PD 81,723 to aid in the protection against ischemia/reperfusion injury, coinciding with a protection against tissue apoptosis, necrosis, and inflammation in dog cardiac tissue, rat hippocampus, and mouse renal tissue just to name a few 38,[45][46][47] . However, there are no studies evaluating the anti-angiogenic potential of PD 81,723. This study is the first to identify PD 81,723 as an angiogenesis inhibitor with validation in vivo with zebrafish and in vitro with HUVECs. While zebrafish and HUVECs both express A 1 receptors 48,49 , A 1 receptors may not be highly expressed in HUVECs 48,50 . It is very likely that the results are mainly due to the allosteric interaction of PD 81,723 at the A 1 receptor, but the possible off target effects of PD 81,723 should be explored further in future work, since binding at the A 1 receptor may not be able to account for the entirety of the results, due to its lower expression.
The discovery and validation of PD 81,723 has answered the question of whether or not an angiogenesis inhibitor could be identified using a fully automated screen of the NIHCCLI&II library with the HTP. PD 81,723 has been identified and validated as an angiogenesis inhibitor in vivo and in vitro; however, further studies are warranted to validate these findings in higher order animals. In addition, a possible switching at the molecular level needs to be investigated in the future, as the HUVECs treated with PD 81,723 appeared to have a slightly different appearance than the controls (see Supplementary Video S2).
The two main takeaways from this work are that a fully automated screen of the NIHCCLI&II library was successfully performed with the HTP, serving as further validation for the methods developed previously 14 , and the discovery of a potentially novel angiogenesis inhibitor from said screen. This is the first fully automated high www.nature.com/scientificreports/ content screen with zebrafish, from embryo dispensing to quantified readout. These results and architecture lay down a foundation that the research community can improve and build on by the creation of even more robust systems and protocols conducive to fully automated experiments that could continue on a trend of minimizing human error and saving time that could be utilized for further innovation. It was a concentration below the concentration that caused a significant result. This was done in hopes of finding a compound that could significantly reduce the endothelial network at a lower concentration and thus be less inclined to be toxic. The drug screening protocol described previously 14 is repeated in triplicate for each working plate. The readout was a count of each pixel, from a 2D confocal image, comprising the endothelial network of a single Tg(kdrl:EGFP) zebrafish embryo affected by a compound in the library. The compounds were introduced to the embryos at 10-11 h post-fertilization (hpf) and the readouts were taken at 4 days post-fertilization (dpf). The pixel count data for each run is saved in an excel file. Values under 4000 are assumed to be dead embryos and excluded. Embryos that are visibly dead prior to imaging and distorted values due to poor images are also removed. Wells that were excluded are set to zero and the corresponding compounds are removed from further analysis. The pixel count values for each compound are divided by the negative control mean value and multiplied by 100 to get a percentage. The percentages for each run are then averaged and ranked. Compounds that reduced the mean endothelial network by 30% or more, compared to normal controls, were considered hits and ranked according to consistency (i.e. a lower standard deviation resulted in a higher ranking). This cut-off percentage was chosen in hopes of finding a compound that performs better than the positive control (I3M at 8 μM), which reduced the endothelial network by an average of 25%. Representative images of the hit compounds, with the pixels containing endothelial cells identified and enhanced, were compared and the 5 compounds that corresponded to the most diminished endothelial network with a relatively normal overall morphology were chosen for further analysis. Fluorescent stereomicroscope imaging. Fluorescent images were taken with a Leica M205 FA stereomicroscope using an EGFP filter [Leica Microsystems Inc., Concord, Ontario Canada] at 1, 2, 3, and 4 dpf to monitor the chemical phenotypes. Unhatched embryos were removed from their chorion manually. The zebrafish were then anesthetised with a 50/50 E2 media/100 ppm clove oil solution and embedded in 2.5% methylcellulose [Sigma-Aldrich, St. Louis, Missouri, USA], a viscous solution that allows the fish to stay in a desired orientation for imaging.

Materials and methods
Confocal imaging. The typical working distance of a high powered objective on a confocal system is less than 0.5 mm and the penetration depth of confocal imaging is roughly in the realm of 300 µm. For this reason, the fish are placed in µ-dishes with a 180 µm thick microscopy plastic bottom [ibidi Gmbh, Munich, Germany] for confocal imaging. The zebrafish are anesthetised with a 50/50 E2 media/100 ppm clove oil solution in the µ-dishes. Most of the solution is removed once the fish are immobilised, leaving solution only in the recessed region of the dishes. Liquefied 1% low melting point agarose is then placed in the dish and the embryo orientation is adjusted as the agarose solidifies. The confocal microscope used for the 3D imaging is a LSM 700 confocal laser scanning microscope [Carl Zeiss Microscopy GmbH, Jena, Germany]. A 5 × objective is used for imaging in which the whole fish is in the field of view. Images are taken throughout the whole depth of visible fluorescence using an EGFP setting with 5 µm between 2D slices and 4 × frame averaging of 512 × 512 frames. A 10 × objective is used for imaging a region of interest. Images are again taken throughout the whole depth of visible fluorescence using an EGFP setting with 2.5 µm between 2D slices and 4 × frame averaging of 512 × 512 frames.
High framerate time-lapse images of Tg(kdrl:EGFP;GATA-1:DsRed) double transgenic zebrafish were taken with a Quorum Spinning Disc Confocal Microscope (Yokogawa X1 head with Borealis) [Quorum Technologies, Puslinch, Ontario, Canada]. The fish were mounted in the same manner as in the LSM 700 imaging. The endothelial tissue was imaged through the whole depth of the visible fluorescence with an EGFP filter and 20 × objective, capturing 100 evenly space 512 × 512 images. This was immediately followed by single plane imaging at the middle cross section of the SIV vessels with a DsRed filter and 20 × objective, capturing 100 images (512 × 512) over 3 s in order to view blood flow. A 3D image of the endothelium was created and a blood flow video, compiled from the 100 time-lapse images, was overlaid on that 3D image with Fiji (open source ImageJ image processing package) 52  Tube formation assay. Growth factor reduced matrigel [BD Biosciences] was plated onto 96-well plates (50 µL/well), incubated at 37 °C for 30 min, and seeded with HUVECs at 10,000 cells/well. Six wells were treated with a control (0.05% DMSO), and six wells were treated with 50 µM PD 81,723. Images from the center of each well were taken with a Nikon Eclipse TS100 inverted microscope [Nikon Instruments Inc., Melville, New York, USA] using a 4 × objective. Six 1 mm 2 sites from the center of each image were analyzed for total capillary tube length at 9 h after seeding. A 3 × 2 grid was created at the center of each image and the length of each branch was measured and summed for each site using the Fiji open source ImageJ image processing package 52 .
Wound healing/cell migration assay. The wound-healing assay methods were adapted from methods described previously 53 . HUVECs were seeded into 12-well plates with 350,000 cells/well and incubated at 37 °C until near 70-80% confluency. A p200 pipet tip was used to scratch a gap across the center of the wells. The cells were treated with a control (0.05% DMSO) or 50 µM PD 81,723 in either complete endothelial growth media (basal media and SingleQuotes bullet kit with 20 ng/ml VEGF-A) [Lonza, Wakersville, Maryland, USA] or endothelial growth media without VEGF-A, with three wells for each condition. Five sites near the center of each gap were imaged with a Zeiss Axio Observer widefield microscope [Carl Zeiss Microscopy GmbH] every 30 min for 24 h at 10 × magnification. The microscope was equipped with an EC Plan-Neofluar objective lens and environmental control (37 °C and 5% CO2 with humidity), using a Hamamatsu ORCA-R2 camera. All of the images were compiled into a video for each site using the Fiji open source ImageJ image processing package 52 and the time of complete wound closure was noted.

Cell proliferation assay. A bromodeoxyuridine (BrdU) incorporation cell proliferation kit [Catalogue #:
2750, Millipore (Canada) Ltd., Etobicoke, Ontario, Canada] was used to look at cell proliferation. HUVECs were treated with a control (0.05% DMSO) or 50 µM PD 81,723 in either complete endothelial growth media or endothelial growth media deficient in VEGF-A, seeding five wells per condition with 20,000 cells in a 96-well plate and subsequently following the manufacturers protocol. Colorimetric optical density (OD) measurements were obtained using a SpectraMax M5e microplate reader [Molecular Devices, San Jose, California, USA] set at 450 nm.
Flow cytometry. The annexin V/PI flow cytometry assay methods were adapted from methods described previously 54 . HUVECs at 60-70% confluency in 6-well plates were treated with 0.05% DMSO or 50 µM PD 81,723 in either complete endothelial growth media or endothelial growth media deficient in VEGF-A for 24 h. www.nature.com/scientificreports/ cate. Immunoblots were conducted as described previously 55 . Cells were lysed in RIPA lysis buffer (10 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 5 mmol/L EDTA; 10 mmol/LNaF; 1 mmol/L phenylmethylsulfonyl fluoride; 1 mmol/L Na3VO4; 50 × protease inhibitor The blots were developed using the ChemiDoc Touch Imaging System [Bio-Rad Laboratories, Hercules, California, USA] and the band intensities were quantified using Image Lab [Bio-Rad]. The mean band intensities for p21, VEGF-A, and AKT were divided by those of the corresponding loading control (β-actin) and p-AKT was divided by the total AKT result. The data was normalized to the corresponding results of 0.05% DMSO treated samples in complete endothelial growth media. Zebrafish. Transgenic Tg(kdrl:EGFP), Tg(fli1:nEGFP), and Tg(GATA-1:DsRed) zebrafish were used in this work. The Tg(kdrl:EGFP) line, also known as Tg(Flk1:EGFP), presents with a vascular endothelial-specific kinase insert domain receptor-like protein (kdrl) promoter, also known as vascular endothelial growth factor receptor 2 (VEGFR-2), directing EGFP expression 56 . EGFP is an enhanced green fluorescence protein with a peak excitation wavelength at 488 nm and a maximum emission wavelength at 509 nm 57,58 . Similarly, the Tg(Fli1:nEGFP) line is a line in which the vascular endothelial-specific friend leukemia integration 1 transcription factor (Fli1) promoter is directing EGFP expression, but in the nucleus 59 . The Tg(GATA-1:DsRed) line is a transgenic line in which the erythroid-specific GATA-binding factor 1 (GATA-1) promoter is directing DsRed expression. GATA-1 is an erythroid transcription factor 60 . DsRed is a red fluorescent protein derived from the coral Discosoma striata with a peak excitation wavelength at 558 nm and a maximum emission wavelength at 583 nm 61 . Tg(kdrl:EGFP) and Tg(GATA-1:DsRed) fish were crossed to create Tg(kdrl:EGFP;GATA-1:DsRed) double transgenic zebrafish.

Compounds.
The Tg(kdrl:EGFP) 56 , Tg(fli1:nEGFP) 59 , and Tg(GATA-1:DsRed) 60 lines were housed in the Li Ka Shing Knowledge Institute (St. Michael's Hospital, Toronto, Ontario, Canada) research vivarium and maintained and staged as previously described 14,62 . All zebrafish experiments were approved by the St. Michael's Hospital Animal Care Committee (Toronto, Ontario, Canada) under protocol ACC867 and performed in compliance with the ARRIVE guidelines. The fish were housed under a 14:10 h light:dark cycle at 28 °C. Embryos were produced by pair mating and raised in 1 × E3 embryo medium (5 mMNaCl, 0.17 mMKCl, 0.33 mMCaCl2, 0.33 mM MgSO4).
HUVECs. Primary HUVECs were isolated from multiple independent donors and maintained as described previously 63,64 . Cells were grown in gelatin coated plates and maintained at 37 °C with 5% CO 2 in a humidified Steri-Cycle CO 2 Incubator [Model 370, Thermo Fisher Scientific]. Experiments were conducted at cell passage 3 or 4. All experiments were performed according to the relevant guidelines and regulations. Statistical analysis. All statistical analyses were performed using a one-way paired ANOVA with a Tukey's post-hoc analysis except for the matrigel tube formation assay results, where an independent samples t-test was used. P-values of less than 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics [IBM, Armonk, New York, USA].

Data availability
The data generated and/or analysed during this work, if not already included in this manuscript, can be obtained from the corresponding author upon request.

Code availability
Details of the automated anti-angiogenesis HTP protocol and the associated programs can be found in previous work 14 . In addition, any code that was developed in the process of performing this work can be obtained from the corresponding author upon request.